Cellular and molecular mechanisms underlying plasma membrane functionality and integrity.

The plasma membrane not only protects the cell from the extracellular environment, acting as a selective barrier, but also regulates cellular events that originate at the cell surface, playing a key role in various biological processes that are essential for the preservation of cell homeostasis. Therefore, elucidation of the mechanisms involved in the maintenance of plasma membrane integrity and functionality is of utmost importance. Cells have developed mechanisms to ensure the quality of proteins that inhabit the cell surface, as well as strategies to cope with injuries inflicted to the plasma membrane. Defects in these mechanisms can lead to the development or onset of several diseases. Despite the importance of these processes, a comprehensive and holistic perspective of plasma membrane quality control is still lacking. To tackle this gap, in this Review, we provide a thorough overview of the mechanisms underlying the identification and targeting of membrane proteins that are to be removed from the cell surface, as well as the membrane repair mechanisms triggered in both physiological and pathological conditions. A better understanding of the mechanisms underlying protein quality control at the plasma membrane can reveal promising and unanticipated targets for the development of innovative therapeutic approaches.

Contribution of extracellular vesicles for the pathogenesis of retinal diseases: shedding light on blood-retinal barrier dysfunction.

Retinal degenerative diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), loom as threats to vision, causing detrimental effects on the structure and function of the retina. Central to understanding these diseases, is the compromised state of the blood-retinal barrier (BRB), an effective barrier that regulates the influx of immune and inflammatory components. Whether BRB breakdown initiates retinal distress, or is a consequence of disease progression, remains enigmatic. Nevertheless, it is an indication of retinal dysfunction and potential vision loss.The intricate intercellular dialogues among retinal cell populations remain unintelligible in the complex retinal milieu, under conditions of inflammation and oxidative stress. The retina, a specialized neural tissue, sustains a ceaseless demand for oxygen and nutrients from two vascular networks. The BRB orchestrates the exchange of molecules and fluids within this specialized region, comprising the inner BRB (iBRB) and the outer BRB (oBRB). Extracellular vesicles (EVs) are small membranous structures, and act as messengers facilitating intercellular communication in this milieu.EVs, both from retinal and peripheral immune cells, increase complexity to BRB dysfunction in DR and AMD. Laden with bioactive cargoes, these EVs can modulate the retinal microenvironment, influencing disease progression. Our review delves into the multifaceted role of EVs in retinal degenerative diseases, elucidating the molecular crosstalk they orchestrate, and their microRNA (miRNA) content. By shedding light on these nanoscale messengers, from their biogenesis, release, to interaction and uptake by target cells, we aim to deepen the comprehension of BRB dysfunction and explore their therapeutic potential, therefore increasing our understanding of DR and AMD pathophysiology.

Cx43-mediated sorting of miRNAs into extracellular vesicles.

Through the exchange of lipids, proteins, and nucleic acids, extracellular vesicles (EV) allow for cell-cell communication across distant cells and tissues to regulate a wide range of physiological and pathological processes. Although some molecular mediators have been discovered, the mechanisms underlying the selective sorting of miRNAs into EV remain elusive. Previous studies demonstrated that connexin43 (Cx43) forms functional channels at the EV surface, mediating the communication with recipient cells. Here, we show that Cx43 participates in the selective sorting of miRNAs into EV through a process that can also involve RNA-binding proteins. We provide evidence that Cx43 can directly bind to specific miRNAs, namely those containing stable secondary structure elements, including miR-133b. Furthermore, Cx43 facilitates the delivery of EV-miRNAs into recipient cells. Phenotypically, we show that Cx43-mediated EV-miRNAs sorting modulates autophagy. Overall, our study ascribes another biological role to Cx43, that is, the selective incorporation of miRNAs into EV, which potentially modulates multiple biological processes in target cells and may have implications for human health and disease.

The good, the bad and the ugly: the impact of extracellular vesicles on the cardiovascular system.

Cardiovascular diseases (CVDs), which encompass a myriad of pathological conditions that affect the heart and/or the blood vessels, remain the major cause of morbidity and mortality worldwide. By transferring a wide variety of bioactive molecules, including proteins and microRNAs (miRNAs), extracellular vesicles (EVs) are recognized as key players in long-range communication across the cardiovascular system. It has been demonstrated that these highly heterogeneous nanosized vesicles participate both in the maintenance of homeostasis of the heart and vessels, and contribute to the pathophysiology of CVDs, thus emerging as promising tools for diagnosis, prognosis and treatment of multiple CVDs. In this review, we highlight the beneficial roles of EV-mediated communication in regulating vascular homeostasis, and inter-organ crosstalk as a potential mechanism controlling systemic metabolic fitness. In addition, the impact of EV secretion in disease development is described, particularly focusing on cardiac remodelling following ischaemia, atherogenesis and atrial fibrillation progression. Finally, we discuss the potential of endogenous and bioengineered EVs as therapeutic tools for CVDs, as well as the suitability of assessing the molecular signature of circulating EVs as a non-invasive predictive marker of CVD onset and progression. This rapidly expanding field of research has established the role of EVs as key conveyors of both cardioprotective and detrimental signals, which might be of relevance in uncovering novel therapeutic targets and biomarkers of CVDs.

Aromatic Plant-Based Functional Foods: A Natural Approach to Manage Cardiovascular Diseases.

Aromatic plants and their essential oils have shown beneficial effects on the cardiovascular system and, therefore, are potential raw materials in the development of functional foods. However, despite their undeniable potential, essential oils present several limitations that need to be addressed, such as stability, poor solubility, undesirable sensory effects, and low bioavailability. The present review provides a current state-of-the-art on the effects of volatile extracts obtained from aromatic plants on the cardiovascular system and focuses on major challenges that need to be addressed to increase their use in food products. Moreover, strategies underway to overcome these limitations are pointed out, thus anticipating a great appreciation of these extracts in the functional food industry.

Alvespimycin Inhibits Heat Shock Protein 90 and Overcomes Imatinib Resistance in Chronic Myeloid Leukemia Cell Lines.

Heat shock protein 90 (HSP90) facilitates folding and stability and prevents the degradation of multiple client proteins. One of these HSP90 clients is BCR-ABL, the oncoprotein characteristic of chronic myeloid leukemia (CML) and the target of tyrosine kinase inhibitors, such as imatinib. Alvespimycin is an HSP90 inhibitor with better pharmacokinetic properties and fewer side effects than other similar drugs, but its role in overcoming imatinib resistance is not yet clarified. This work studied the therapeutic potential of alvespimycin in imatinib-sensitive (K562) and imatinib-resistant (K562-RC and K562-RD) CML cell lines. Metabolic activity was determined by the resazurin assay. Cell death, caspase activity, mitochondrial membrane potential, and cell cycle were evaluated by means of flow cytometry. Cell death was also analyzed by optical microscopy. HSPs expression levels were assessed by western blotting. Alvespimycin reduced metabolic activity in a time-, dose-, and cell line-dependent manner. Resistant cells were more sensitive to alvespimycin with an IC50 of 31 nM for K562-RC and 44 nM for K562-RD, compared to 50 nM for K562. This drug induced apoptosis via the mitochondrial pathway. In K562 cells, alvespimycin induced cell cycle arrest in G0/G1. As a marker of HSP90 inhibition, a significant increase in HSP70 expression was observed. Our results suggest that alvespimycin might be a new therapeutic approach to CML treatment, even in cases of resistance to imatinib.

Osteosarcoma-Derived Exosomes as Potential PET Imaging Nanocarriers for Lung Metastasis.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected with current clinical tools. Herein, an exosome (EX)-based imaging tool is developed for lung micrometastasis by positron emission tomography (PET) using osteosarcoma-derived EXs as natural nanocarriers of the positron-emitter copper-64 (64 Cu). Exosomes are isolated from metastatic osteosarcoma cells and functionalized with the macrocyclic chelator NODAGA for complexation with 64 Cu. Surface functionalization has no effect on the physicochemical properties of EXs, or affinity for donor cells and endows them with favorable pharmacokinetics for in vivo studies. Whole-body PET/magnetic resonance imaging (MRI) images in xenografted models show a specific accumulation of 64 Cu-NODAGA-EXs in metastatic lesions as small as 2-3 mm or in a primary tumor, demonstrating the exquisite tropism of EXs for homotypic donor cells. The targetability for lung metastasis is also observed by optical imaging using indocyanine green (ICG)-labeled EXs and D-luciferin-loaded EXs. These findings show that tumor-derived EXs hold great potential as targeted imaging agents for the noninvasive detection of small lung metastasis by PET. This represents a step forward in the biomedical application of EXs in imaging diagnosis with increased translational potential.

EHD1 Modulates Cx43 Gap Junction Remodeling Associated With Cardiac Diseases.

RATIONALE: Efficient communication between heart cells is vital to ensure the anisotropic propagation of electrical impulses, a function mainly accomplished by gap junctions (GJ) composed of Cx43 (connexin 43). Although the molecular mechanisms remain unclear, altered distribution and function of gap junctions have been associated with acute myocardial infarction and heart failure. OBJECTIVE: A recent proteomic study from our laboratory identified EHD1 (Eps15 [endocytic adaptor epidermal growth factor receptor substrate 15] homology domain-containing protein 1) as a novel interactor of Cx43 in the heart. METHODS AND RESULTS: In the present work, we demonstrate that knockdown of EHD1 impaired the internalization of Cx43, preserving gap junction-intercellular coupling in cardiomyocytes. Interaction of Cx43 with EHD1 was mediated by Eps15 and promoted by phosphorylation and ubiquitination of Cx43. Overexpression of wild-type EHD1 accelerated internalization of Cx43 and exacerbated ischemia-induced lateralization of Cx43 in isolated adult cardiomyocytes. In addition, we show that EHDs associate with Cx43 in human and murine failing hearts. CONCLUSIONS: Overall, we identified EHDs as novel regulators of endocytic trafficking of Cx43, participating in the pathological remodeling of gap junctions, paving the way to innovative therapeutic strategies aiming at preserving intercellular communication in the heart.

1,8-Cineole ameliorates right ventricle dysfunction associated with pulmonary arterial hypertension by restoring connexin43 and mitochondrial homeostasis.

For the first time, the present study unravels a cardiospecific therapeutic approach for Pulmonary Arterial Hypertension (PAH), a disease with a very poor prognosis and high mortality rates due to right ventricle (RV) dysfunction. We first established a new in vitro model of high-pressure-induced hypertrophy that closely resembles heart defects associated with PAH and validated our in vitro findings on a preclinical in vivo model of monocrotaline (MCT)-induced PAH. Our results showed the in vitro antihypertrophic effect of 1,8-cineole, a monoterpene widely found in several essential oils. Also, a decrease in RV hypertrophy and fibrosis, and an improvement in heart function in vivo was observed, when 1,8-cineole was applied topically. Furthermore, 1,8-cineole restored gap junction protein connexin43 distribution at the intercalated disks and mitochondrial functionality, suggesting it may act by preserving cardiac cell-to-cell communication and bioenergetics. Overall, our results point out a promising therapeutic compound that can be easily applied topically, thus paving the way for the development of effective cardiac-specific therapies to greatly improve PAH outcomes.

The Role of Essential Oils and Their Main Compounds in the Management of Cardiovascular Disease Risk Factors.

Cardiovascular diseases (CVDs) are a global health burden that greatly impact patient quality of life and account for a huge number of deaths worldwide. Despite current therapies, several side effects have been reported that compromise patient adherence; thus, affecting therapeutic benefits. In this context, plant metabolites, namely volatile extracts and compounds, have emerged as promising therapeutic agents. Indeed, these compounds, in addition to having beneficial bioactivities, are generally more amenable and present less side effects, allowing better patient tolerance. The present review is an updated compilation of the studies carried out in the last 20 years on the beneficial potential of essential oils, and their compounds, against major risk factors of CVDs. Overall, these metabolites show beneficial potential through a direct effect on these risk factors, namely hypertension, dyslipidemia and diabetes, or by acting on related targets, or exerting general cellular protection. In general, monoterpenic compounds are the most studied regarding hypotensive and anti-dyslipidemic/antidiabetic properties, whereas phenylpropanoids are very effective at avoiding platelet aggregation. Despite the number of studies performed, clinical trials are sparse and several aspects related to essential oil's features, namely volatility and chemical variability, need to be considered in order to guarantee their efficacy in a clinical setting.

Targeting mitochondrial fusion and fission proteins for cardioprotection.

New treatments are needed to protect the myocardium against the detrimental effects of acute ischaemia/reperfusion (IR) injury following an acute myocardial infarction (AMI), in order to limit myocardial infarct (MI) size, preserve cardiac function and prevent the onset of heart failure (HF). Given the critical role of mitochondria in energy production for cardiac contractile function, prevention of mitochondrial dysfunction during acute myocardial IRI may provide novel cardioprotective strategies. In this regard, the mitochondrial fusion and fissions proteins, which regulate changes in mitochondrial morphology, are known to impact on mitochondrial quality control by modulating mitochondrial biogenesis, mitophagy and the mitochondrial unfolded protein response. In this article, we review how targeting these inter-related processes may provide novel treatment targets and new therapeutic strategies for reducing MI size, preventing the onset of HF following AMI.

Mitochondrial ion channels as targets for cardioprotection.

Acute myocardial infarction (AMI) and the heart failure (HF) that often result remain the leading causes of death and disability worldwide. As such, new therapeutic targets need to be discovered to protect the myocardium against acute ischaemia/reperfusion (I/R) injury in order to reduce myocardial infarct (MI) size, preserve left ventricular function and prevent the onset of HF. Mitochondrial dysfunction during acute I/R injury is a critical determinant of cell death following AMI, and therefore, ion channels in the inner mitochondrial membrane, which are known to influence cell death and survival, provide potential therapeutic targets for cardioprotection. In this article, we review the role of mitochondrial ion channels, which are known to modulate susceptibility to acute myocardial I/R injury, and we explore their potential roles as therapeutic targets for reducing MI size and preventing HF following AMI.

Exosomes derived from microglia exposed to elevated pressure amplify the neuroinflammatory response in retinal cells.

Glaucoma is a degenerative disease that causes irreversible loss of vision and is characterized by retinal ganglion cell (RGC) loss. Others and we have demonstrated that chronic neuroinflammation mediated by reactive microglial cells plays a role in glaucomatous pathology. Exosomes are extracellular vesicles released by most cells, including microglia, that mediate intercellular communication. The role of microglial exosomes in glaucomatous degeneration remains unknown. Taking the prominent role of microglial exosomes in brain neurodegenerative diseases, we studied the contribution of microglial-derived exosomes to the inflammatory response in experimental glaucoma. Microglial cells were exposed to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure, the main risk factor for glaucoma. Naïve microglia (BV-2 cells or retinal microglia) were exposed to exosomes derived from BV-2 cells under EHP conditions (BV-Exo-EHP) or cultured in control pressure (BV-Exo-Control). We found that BV-Exo-EHP increased the production of pro-inflammatory cytokines, promoted retinal microglia motility, phagocytic efficiency, and proliferation. Furthermore, the incubation of primary retinal neural cell cultures with BV-Exo-EHP increased cell death and the production of reactive oxygen species. Exosomes derived from retinal microglia (MG-Exo-Control or MG-Exo-EHP) were injected in the vitreous of C57BL/6J mice. MG-Exo-EHP sustained activation of retinal microglia, mediated cell death, and impacted RGC number. Herein, we show that exosomes derived from retinal microglia have an autocrine function and propagate the inflammatory signal in conditions of elevated pressure, contributing to retinal degeneration in glaucomatous conditions.

Caveolin-1 Modulation Increases Efficacy of a Galacto-Conjugated Phthalocyanine in Bladder Cancer Cells Resistant to Photodynamic Therapy.

Photodynamic therapy (PDT) has demonstrated encouraging anticancer therapeutic results, but the current clinically approved photosensitizers (PSs) are not ideal in the treatment of bladder cancer. Conventional PSs have low selectivity to the bladder tumor tissue and induce toxicity or bystander effects on nontumor urothelium. Previous studies demonstrated that the use of galactose-photosensitizer (PS) conjugates is a more selective method of delivering PDT-mediated toxicity due to their ability to recognize carbohydrate-binding domains overexpressed in bladder tumors. Using patient-derived bladder tumor specimens cultured ex vivo and bladder cancer cell lines with different PDT sensitivity, we find that a galactose-phthalocyanine (PcGal16) accumulates in bladder tumors expressing galactose-binding proteins and internalizes through an endocytic process. The endocytosis mechanism is cell line-dependent. In HT-1376 bladder cancer lines resistant to PDT, depletion of caveolin-1-the main structural protein of caveolae structures-increased the amount of sugar-binding proteins, i.e. GLUT1, at the cell membrane resulting in an improved PcGal16 uptake and PDT efficacy. These data show the potential of ex vivo cultures of bladder cancer, that ideally could mimic the original microenvironment, in screening galacto-PDT agents. Additionally, our studies demonstrate that PDT efficacy in bladder cancer depends on the endocytic mechanisms that regulate PS accumulation and internalization in cancer cells.

The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication.

Given the low mitotic activity of cardiomyocytes, the contractile unit of the heart, these cells strongly rely on efficient and highly regulated mechanisms of protein degradation to eliminate unwanted potentially toxic proteins. This is particularly important in the context of disease, where an impairment of protein quality control mechanisms underlies the onset and development of diverse cardiovascular maladies. One of the biological processes which is tightly regulated by proteolysis mechanisms is intercellular communication. The different types of cells that form the heart, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, can communicate directly, through gap junctions (GJ) or tunneling nanotubes (TNT), or at long distances, via extracellular vesicles (EV) or soluble factors.The direct communication between cardiomyocytes is vital to ensure the anisotropic propagation of the electrical impulse, which allows the heart to beat in a coordinated and synchronized manner, as a functional syncytium. The rapid and efficient propagation of the depolarization wave is mainly conducted by low resistance channels called GJ, formed by six subunits of a family of proteins named Cxs. Dysfunctional GJ intercellular communication, due to increased degradation and/or redistribution of connexin43 (Cx43), the main Cx present in the heart, has been associated with several cardiac disorders, such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure. Besides electrical coupling, a fine-tuned exchange of information, namely proteins and microRNAs, conveyed by EV is important to ensure organ function and homeostasis. Disease-induced deregulation of EV-mediated communication between cardiac cells has been implicated in diverse processes such as inflammation, angiogenesis, and fibrosis. Therefore, a better understanding of the mechanisms whereby proteolysis modulates the cross talk between cardiac cells is of utmost importance to develop new strategies to tackle diseases caused by defects in intercellular communication.

Ischaemia alters the effects of cardiomyocyte-derived extracellular vesicles on macrophage activation.

Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell-cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte-derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro-inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL-1ß and IL-6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL-1ß, IL-6, IL-10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum-circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte-derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post-infarction remodelling.

Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes.

Communication is important to ensure the correct and efficient flow of information, which is required to sustain active social networks. A fine-tuned communication between cells is vital to maintain the homeostasis and function of multicellular or unicellular organisms in a community environment. Although there are different levels of complexity, intercellular communication, in prokaryotes to mammalians, can occur through secreted molecules (either soluble or encapsulated in vesicles), tubular structures connecting close cells or intercellular channels that link the cytoplasm of adjacent cells. In mammals, these different types of communication serve different purposes, may involve distinct factors and are mediated by extracellular vesicles, tunnelling nanotubes or gap junctions. Recent studies have shown that connexin 43 (Cx43, also known as GJA1), a transmembrane protein initially described as a gap junction protein, participates in all these forms of communication; this emphasizes the concept of adopting strategies to maximize the potential of available resources by reutilizing the same factor in different scenarios. In this Review, we provide an overview of the most recent advances regarding the role of Cx43 in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions.

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.